Human diacylglycerol kinase β (HDAGKβ) protein and nucleotide sequences encoding the same

ABSTRACT

The present invention relates to human diacylglycerol kinase proteins (hDAGK) and particularly to human diacylglycerol kinase β (hDAGKβ) protein, and to related nucleotide sequences, expression vectors, cell lines, antibodies, screening methods, compounds, methods of production and methods of treatment, as well as other related aspects.

This application is filed pursuant to 35 U.S.C. §371 as a United StatesNational Phase Application of International Application No.PCT/GB99/04421 filed Dec. 23, 1999, which claims priority fromGB9903430.8 filed Feb. 15, 1999.

FIELD OF THE INVENTION

The present invention relates to human diacylglycerol kinase proteins(hDAGK) and particularly to human diacylglycerol kinase β (hDAGKβ)protein, and to related nucleotide sequences, expression vectors, celllines, antibodies, screening methods, compounds, methods of productionand methods of treatment, as well as other related aspects.

BACKGROUND OF THE INVENTION

Diacylglycerol kinases (DAGKs) are a family of enzymes that convertdiacylglycerol (DAG) to phosphatidic acid and are therefore known toattenuate DAG-dependent protein kinase C activation (PCK) (1).

Five types of DAGKs have been described. Type I DAGKs contains fourconserved regions, the N-terminal region (C 1), two sets of EF-handmotifs (C2), two cysteine-rich zinc finger like structures (C3) and theC-terminal region (C4). Type II isoenzymes contain a pleckstrin homology(PH) domain at the N-terminus. Type Ill contains only the zinc finger(C3) and the catalytic region (C4). Type IV contains four ankyrinrepeats near the carboxyl terminus. Type V contains three instead of twozinc finger structures, a proline-rich region and a PH domain with anoverlapping Ras-associating (RA) domain. All share two domains, the C2and the C3 (2).

A 90 kDa DAGKβ (3) belonging to type I was found by screening a ratbrain cDNA library using fragments of rat a DAGK cDNA under lowstringency conditions. The cDNA clone obtained was completely sequenced.The rat DAGKβ cDNA has an open reading frame of 5927 nucleotides andencodes for a protein of 801 amino acids with a predicted relativemolecular mass of 90,000. Analysis of the amino acid sequence identified2 EF-hand motifs (aa 152-180 and 197-225), of two cysteine-richzinc-finger-like sequences (aa 257-292 and 319-356), and putativeATP-binding sites (aa 266-294 and 533-560).

Brain expression has been described for the known DAGK isoforms (2),which include DAGKα, DAGKη, DAGKζ and DAGKθ. A particular distributionrestricted to specific regions of the central nervous system (CNS) wasdescribed for the DAGKβ form, originally identified in the rat (90 DaDAGKβ). The rat beta form is predominantly localised in neurons of thecaudate-putamen, the accubens nucleus and the olfactory tubercle. Suchbrain regions are among the main CNS dopaminergic, serotonergic,acetylcholinergic and glutamatergic terminal fields (4).

It has also been demonstrated that some metabotropic dopamine,serotonin, glutamate, acetylcholine and several peptide receptors arecoupled with the phosphoinositide signal transduction system (5).Lithium is known as one of the most effective therapies for bipolardisorders. Although the biological mechanisms of the mood stabilisingproperties of lithium are not well understood, experimental evidenceindicates that lithium modulates the phosphoinositide signaltransduction system (6) by inhibiting the phosphatase that liberatesinositol from inositol phosphate (IP), and by modifying the activity ofthe phospholipase C (PLC)-dependent signalling pathways, including thelevels of the second messenger diacylglycerol (DAG) that activatesprotein kinase C (PKC).

With this background in mind, the present inventors have determined thatthe DAGKs, and particularly the human ortholog of rat 90 kDa DAGKβ(hDAGKβ) and variants thereof will provide targets for the developmentof novel mood stabilising agents and therapeutic agents for treatment ofother disorders.

Clearly there is a need for proteins and related nucleotide sequencesthat may be used to screen for mood stabilising agents and which mayalso play a role in preventing, ameliorating or correcting mooddisorders and dysfunction and other neurological diseases.

Accordingly, it is an object of the present invention to identifytargets for screening of novel therapeutic agents. It is another objectof the invention to locate and characterise human DAGKβ and variantsthereof. Other objects of the present invention will become apparentfrom the following detailed description thereof.

SUMMARY OF THE INVENTION

According to one embodiment of the present invention there is providedan isolated human diacylglycerol kinase β (hDAGKβ) protein or a variantthereof. In a particularly preferred embodiment of this invention thehuman diacylglycerol kinase β (hDAGKβ) has the amino acid sequence setout in Seq ID No 1 or Seq ID No 4. The hDAGKβ protein having the aminoacid sequence set out in Seq ID No 4 is referred to as SV-hDAGKβprotein.

According to one embodiment of the present invention there is provided anucleotide sequence encoding a human diacylglycerol kinase β (hDAGKβ)protein or a variant thereof, or a nucleotide sequence that iscomplementary thereto. In a particular preferred embodiment of theinvention the polynucleotide comprises the sequence set out in Seq ID No3 or Seq ID No 6. The hDAGKβ protein having the polinucletide sequenceset out in Seq ID No 6 is referred to as SV-hDAGKβ protein.

In accordance with another aspect of the invention there is provided anexpression vector comprising a nucleic acid sequence as referred toabove which is capable of expressing a hDAGKβ protein.

According to a further aspect of the invention there is provided astable cell line comprising an expression vector as referred to above.Preferably the cell line is a modified HEK293T, CHO, HeLa, Sf9 or COScell line.

According to yet a further aspect of the invention there is provided anantibody specific for a hDAGKβ protein.

According to still another aspect of the invention there is provided amethod for identification of a compound that exhibits DAGK modulatingactivity, comprising contacting a DAGK protein with a test compound anddetecting modulation of enzyme activity or detecting enzyme inactivity.Preferably the DAGK is hDAGKβ or a variant thereof.

According to another aspect of the invention there is provided acompound which modulates hDAGK activity, identifiable by the methodreferred to above. Preferred compounds according to the presentinvention are those that modulate hDAGKβ activity or a variant thereof.

According to another aspect of the invention there is provided acompound that modulates hDAGK activity. Preferred compounds according tothe present invention are those that modulate hDAGKβ activity or avariant thereof.

According to a further aspect of the invention there is provided amethod of treatment or prophylaxis of a disorder that is responsive tomodulation of hDAGK activity in a human patient, which comprisesadministering to said patient an effective amount of a compound asreferred to above. Conveniently the hDAGK is hDAGKβ or a variantthereof. Preferably the disorder is a mood disorder, epilepsy, aneurodegenerative disorder, anxiety, schizophrenia, migraine, drugdependence, stroke, Alzheimer's dementia or Parkinson's disease.

According to a further aspect of the invention there is provided amethod of treatment or prophylaxis of a disorder that is responsive tomodulation of hDAGK activity in a human patient which comprisesadministering to said patient an effective amount of a modulator ofhDAGK activity. Conveniently the hDAGK is hDAGKβ or a variant thereof.Preferably the disorder is a mood disorder, epilepsy, aneurodegenerative disorder, anxiety, schizophrenia, migraine, drugdependence, stroke, Alzheimer's dementia or Parkinson's disease.

According to another aspect of the invention there is provided use of acompound as referred to above in a method of formulating a medicamentfor treatment or prophylaxis of a disorder that is responsive tomodulation of hDAGK activity in a human patient. Conveniently the hDAGKis hDAGKβ or a variant thereof. Preferably the disorder is a mooddisorder, epilepsy, a neurodegenerative disorder, anxiety,schizophrenia, migraine, drug dependence, stroke, Alzheimer's dementiaor Parkinson's disease.

According to another aspect of the invention there is provided use of amodulator of hDAGK activity in a method of formulating a medicament fortreatment or prophylaxis of a disorder that is responsive to modulationof hDAGK activity in a human patient. Conveniently the hDAGK is hDAGKβor a variant thereof. Preferably the disorder is a mood disorder,epilepsy, a neurodegenerative disorder, anxiety, schizophrenia,migraine, drug dependence, stroke, Alzheimer's dementia or Parkinson'sdisease.

According to another aspect of the invention there is provided a methodof producing a hDAGKβ protein or a variant thereof comprisingintroducing into an appropriate cell line a suitable vector comprising anucleotide sequence encoding for a hDAGKβ protein or a variant thereof,under conditions suitable for obtaining expression of the hDAGKβ proteinor variant.

Seq ID No 3 shows the complete nucleotide sequence of the human DAGKβ.Seq ID No 5 shows the complete nucleotide sequence of the SV-hDAGKβ.

Seq ID No 1 shows the nucleotide and encoded amino acid sequence of thehuman DAGKβ sequence. Seq ID No 4 shows the nucleotide and encoded aminoacid sequence of the SV-hDAGKβ.

Seq ID No 7 shows pairwise alignment of hDAGKβ and SV-hDAGKβ full-lengthamino acids sequences with rat homologue.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings are illustrative of embodiments of the inventionand are not meant to limit the scope of the invention as encompassed bythe claims.

FIGS. 1A and 1B shows a reverse transcriptase polymerase chain reaction(RT-PCR) of human adult and human foetal brain (polyA+RNA).

FIGS. 2A and 2B show the position of the alternatively spliced exonsthat generate a family of hDAGKβ transcripts. The alternatively splicedexons are illustrated as white boxes. The dashed box identifies theSV-DAGKβ transcripts. Panel A: hDAGKβ transcripts generated throughalternative splicing. Panel B: SV-hDAGKβ transcripts generated throughalternative splicing.

FIG. 3 shows RT-PCR analysis of the expression of hDAGKβ and SV-hDAGKβin human tissue cDNAs. PCR reactions were carried out separately foreach primer pair (hDAGKβ, SV-hDAGKβ and β-actin control) on theindicated tissue cDNAs, and reactions corresponding to the same tissuetemplate were loaded in the same well.

FIG. 4 shows RT-PCR analysis of hDAGKβ and SV-hDAGKβ expression innormal and Alzheimer cerebellum. β-actin was used as a control of RNAlevels in the samples.

DETAILED DESCRIPTION OF THE INVENTION

Throughout the present specification and the accompanying claims thewords “comprise” and “include” and variations such as “comprises”,“comprising”, “includes” and “including” are to be interpretedinclusively. That is, these words are intended to convey the possibleinclusion of other elements or integers not specifically recited, wherethe context allows.

As referred to above, the present invention relates to humandiacylglycerol kinase β (hDAGKβ) protein, sequence information for whichis provided in Seq ID No 1 or Seq ID No 4. In the context of thisinvention the term “isolated” is intended to convey that the protein isnot in its native state, insofar as it has been purified at least tosome extent or has been synthetically produced, for example byrecombinant methods. The term “isolated” therefore includes thepossibility of the protein being in combination with other biological ornon-biological material, such as cells, suspensions of cells or cellfragments, proteins, peptides, organic or inorganic solvents, or othermaterials where appropriate, but excludes the situation where theprotein is in a state as found in nature.

Routine methods can be employed to purify and/or synthesise the proteinsaccording to the invention. Such methods are well understood by personsskilled in the art, and include techniques such as those disclosed inSambrook et al. (7), the disclosure of which is included herein in itsentirety by way of reference.

By the term “variant” what is meant throughout the specification andclaims is that other peptides or proteins which retain the sameessential character of the diacylglycerol kinase for which sequenceinformation is provided, are also intended to be included within thescope of the invention. For example, other peptides or proteins withgreater than about 80%, preferably at least 90% and particularlypreferably at least 95% homology with the sequences provided areconsidered as variants of the enzymes. Such variants may include thedeletion, modification or addition of single amino acids or groups ofamino acids within the protein sequence, as long as the peptidemaintains the biological functionality of a hDAGKβ. The rat DAGKβprotein is of course excluded from the definition of “variant”.

Human DAGKβ is expressed in human brain and has the highest (˜95%identity) sequence homology with the rat DAGKβ. Therefore, hDAGKβ islikely to be the human orthologue of rat DAGKβ. Seq ID No 1 reveals a3926 bp (base pair) open reading frame which encodes an 804 amino acidprotein. This deduced protein sequence is ˜95% identical to the ratDAGKβ and shares many of its characteristics and all the domains.

The invention also includes nucleotide sequences identified as Seq ID No1 or Seq ID No 4 that encode for hDAGKβ protein or variants thereof aswell as nucleotide sequences that are complementary thereto. Preferablythe nucleotide sequence is a DNA sequence and most preferably, a cDNAsequence. Such nucleotides can be isolated or synthesised according tomethods well know in Sambrook et al. (7), the disclosure of which isincluded herein in its entirety by way of reference.

The present invention also includes expression vectors that comprisenucleotide sequences encoding for the hDAGKβ protein or variantsthereof. A further aspect of the invention relates to an expressionvector comprising nucleotide sequences encoding for hDAGKβ protein orvariants thereof. Such expression vectors are routinely constructed inthe art of molecular biology and may for example involve the use ofplasmid DNA and appropriate initiators, promoters, enhancers and otherelements, such as for example polyadenylation signals which may benecessary, and which are positioned in the correct orientation, in orderto allow for protein expression. Suitable vectors would be apparent topersons skilled in the art. By way of further example in this regard werefer to (7), the disclosure of which is included herein in itsentirety.

The invention also includes cell lines that have been modified toexpress the novel protein and variants thereof. Such cell lines includetransient, or preferably stable higher eukaryotic cell lines, such asmammalian cells or insect cells, lower eukaryotic cells, such as yeastor prokaryotic cells such as bacterial cells. Particular. examples ofcells that have been modified by insertion of vectors encoding for theproteins according to the invention include the mammalian HEK293T, CHO,HeLa, Sf9 and COS cells.

It is also possible for the protein and variants thereof of theinvention to be transiently expressed in a cell line, such as forexample in a baculovirus expression or in an E. coli system. Suchsystems, which are adapted to express the proteins according to theinvention, are also included within the scope of the present invention.

According to another aspect, the present invention relates to antibodies(either polyclonal or preferably monoclonal antibodies) which have beenraised by standard techniques and are specific for the protein orvariants thereof according to the invention. Such antibodies could forexample, be useful in purification, isolation or screening involvingimmuno-precipitation techniques and may be used as tools to furtherelucidate protein function, or indeed as therapeutic agents in their ownright. Antibodies may also be raised against specific epitopes of theproteins according to the invention.

An important aspect of the present invention is the use of hDAGKproteins in screening methods designed to identify compounds which actas enzyme ligands and which may be useful as modulators of enzymaticactivity. In general terms, such screening methods will involvecontacting the hDAGK protein concerned, which may be any known or as yetunrecognised hDAGK protein or variant thereof, preferably hDAGKβ, with atest compound and then detecting modulation of the enzymatic activity,or indeed detecting enzyme inactivity, which results.

The present invention also includes within its scope those compounds,which are identified as possessing useful hDAGK modulation activity.Such activity can be determined by the screening methods referred toabove. The screening methods comprehended by the invention are generallywell known to those skilled in the art. An example of such an approachis provided in the experimental section of this specification.

Another aspect of the present invention is the use of compounds whichhave been identified by screening techniques referred to above, or othercompounds found to exhibit hDAGK modulating activity, in the treatmentor prophylaxis of disorders that are responsive to modulation of a hDAGKactivity, particularly hDAGKβ activity, in a human patient. By the term“modulation” what is meant is that there will be either agonism orantagonism of the enzymatic activity, which results from ligand bindingof the compound at the catalytic or regulatory sites of the hDAGKprotein. These proteins have been implicated in disorders of the centralnervous system (CNS), and therefore, modulation of hDAGK enzymaticactivity in these tissues will result in a positive therapeutic outcomein relation to such disorders.

In particular, the compounds which will be identified using thescreening techniques according to the invention will have utility fortreatment and/or prophylaxis of disorders such as mood disorders,epilepsy, anxiety, schizophrenia, drug dependence, neurodegenerativedisorders. Some specific examples of disorders which may be treated orprevented by administration of compounds identified in the screeningtechniques according to the present invention are unipolar and bipolardepression, stroke, Alzheimer's dementia, Parkinson's disease, smokingcessation, and ethanol, nicotine, cocaine and heroine abuse. It is to beunderstood, however, that the mention of such disorders is by way ofexample only, and is not intended to be limiting on the scope of theinvention.

The compounds which are identified according to the screening methodsoutlined above may be formulated with standard pharmaceuticallyacceptable carriers and/or excipients, as is routine in thepharmaceutical art, and as is fully described in Remmington'sPharmaceutical Sciences, Mack Publishing Company, Eastern Pa., 17th Ed,1985; the disclosure of which is included herein in its entirety by wayof reference.

The compounds may be administered via enteral or parenteral routes suchas via oral, buccal, anal, pulmonary, intravenous, intraarterial,intramuscular, intraperitoneal, topical or other appropriateadministration routes.

It will further be appreciated that the amount of a compound of theinvention required for use in treatment will vary with the nature of thecondition being treated, the route of administration and the age, sex,weight and general condition of the patient, and will ultimately be atthe discretion of the attendant physician. In general, however, dosesemployed for adult human treatment will typically be in the range ofbetween about 2 mg to about 800 mg per day.

The present invention will be further explained, by way of example, inthe following experimental section.

Experimental

Identification of Human Genomic Sequences Corresponding to BipolarPatients' ESTs Obtained by in Silico Analysis

An extensive search using keywords in both public (GenBank) and private(Incyte) databases resulted in some ESTs annotated as specific forbipolar diseases. Most of GenBank ESTs referred to the StanleyNeurovirology Laboratory (John Hopkins School of Medicine, Baltimore),where they were obtained by subtractive hybridisation of frontal cortexRNA from individuals with bipolar disorder and individuals withoutpsychiatric diseases as controls.

A first comparison against dbEST (8) using the Blastn (9) alignmentprogram revealed that most of the bipolar specific ESTs do notcorrespond to any sequence of known function and no other overlappingESTs can be found to create contigs and enlarge the sequences.Therefore, in a preliminary analysis only those presenting enoughinformation to proceed with an in silico work were considered, inparticular those ESTs that showed 100% identity with a genomic sequence.The. genomic sequence identified for the EST-S4 (GenBank acc. n.AF019352) is an unannotated 172-Kb length sequence (GenBank acc. n.AC005039) consisting of 2 contigs, for which the order is not known,interrupted by an N bases gap.

A detailed analysis was performed on the whole uncharacterised regionwith the aim of extracting all the high complexity sub-regions thatusually contain coding sequences. The low complexity and highlyredundant regions found in portions of the sequence were isolated usingthe SEG (segment sequences by local complexity) tool (10, 11,) andcompared against all the available sequence databases to exclude anypossible translation. As expected, these low complexity regionscorresponded mainly to different families of ALU sequences thereforewere masked in the subsequent gene prediction approach.

The EST-S4 Belongs to a Novel Human Gene Orthologue to the Rat DAGKβgene

The Blastx alignment program was used to compare the 4 Kb genomic regionsurrounding the EST-S4 region with the amino acid sequences of SwissProtand TREMBL databases. Only some local similarities with a highstatistical significance with the rat DAGKβ gene were evidenced andsuggest an authentic relationship. To verify if this result wasconsistent with a possible gene construct an in silico exon trappingmethod (12) was applied on the AC005039 sequence. GeneMark (13), Xpound(12) and GRAIL (14) exon prediction tools were used to locate thepotential coding regions within AC005039. Three exons (a, b and c) werefound to be consistent with the same ORF and to correspond to exons 22,23 and 24 of the rat DAGKβ gene. The region was 32 Kb wide and containedthe EST-S4 sequence.

Searching the more recent set of public domain nucleotide sequences (NewGenBank updates), a partial mRNA sequence of 3742 bp from a human adultbrain (KIAA0718, Acc. No. AB018261) was found to partially overlap the 4kb genomic sequence (100% identity with the three exons so faridentified). The predicted protein sequence (defined in GenPept asKIAA0718 protein) was limited to 742 aa with a N-terminal truncationdescribed. Homology search in protein databases indicates a highsimilarity with rat DAGKβ.

Chromosomal Localisation of the Putative hDAGKβ

The genomic sequence AC005039 is annotated as an unfinished sequencemapped on chromosome 7. An “In silico” STS (Sequence Tagged Site)content analysis (15) was performed on the sequence, and the 3 STSs(sWSS2950, D7S2174 and sWSS2190) found in the sequence confirmed thatthe hDAGKβ is localised on 7p21.

In silico Cloning of Full-length of the hDAGKβ mRNA

Comparing (tBlastn) the first two exons of the rat protein against theGenBank HTG (high throughput genomic) sequences database, two furtheroverlapping genomic sequences (100% identity) were identified (GenBankacc. n. H_GS120K9 and AC006045). These sequences do not overlap withAC005039 but both contain four STSs (sWSS3226, sWSS822, sWSS2758, andsWSS2091) and belong to the same YAC clone (CEPH791G01) where AC005039is located. These data indicate that the exons identified belong to thehDAGKβ gene and provide the information missing in the KIAA0718structure to complete the full length sequence of the predicted protein

Identification and Characterisation of Human DAGKβ Variants in cDNALibraries

Three DNA probes were used to screen a human foetal brain cDNA library(cat. n. 936206 Stratagene, La Jolla, Calif.). The 246 bp fragment wasobtained by reverse transcriptase polymerase chain reaction (RT-PCR)(16) with the oligo pairs previously described (see above paragraph),and used for radioactive hybridisation. The two primer pairsDAGKIAAfor/DAGKIAArev (5′TGAAGACATTCCTGGAAGCC, 5′GACTGTGTACTTGCAGAAGG),and 5hDAGKfor/5hDAGKrev (5′CCATGACAAACCAGGAAAAATGG,5′GATTATACTTTGCAAGCACACC) were used to obtain 2 RT-PCR products of 647bp and of 151 bp respectively from foetal brain polyA+RNA (Clontech)(FIG. 1, panel B). The PCR conditions included an initial hot-start at94° C. for 2 minutes, followed by 35 cycles at 94° C. for 1 minute, 56°C. (DAGKIAAfor/DAGKIAArev) or 58° C. (5hDAGKfor/5hDAGKrev) for 1 minute,72° C. for 1 minute and terminated by 7 minutes at 72° C. The resultingPCR amplicons were separated on a 2% agarose gel and used forradioactive hybridisation.

To fully sequence the cDNA clones isolated, the library inserts werefirst subcloned in pBluescript KS vector (Stratagene, La Jolla, Calif.).After transformation, colonies were screened by hybridisation with thepreviously described probes. Positive colonies were subjected to directsequencing (17) using the T3 and T7 primers. The DNA sequences obtainedwere assembled using the GCG package, translated and aligned with therat DAGKβ gene using CLUSTAL (18). Two positive clones were isolatedusing a human DAGKβ-specific probe covering sequences from position 1524to position 2360 and their sequence covered by sequencing on both DNAstrands. The clones contained the last three exons and part of the 3′untranslated region (UTR) of the hDAGKβ sequence.

The tissue distribution of hDAGKβ gene was established by radioactivehybridisation on multi-Tissue northern blots (Clontech) according to themanufacturer's recommendations. The probes were obtained by RT-PCRamplification of different portions of the coding region of hDAGKβincluding the 3′ splice variant specific probe.

Identification, Characterisation and Cloning of the 3′ End of SV-DAGKβSplice Variant

The GCG package (Wisconsin Package Version 9.0, Genetics Computer Group(GCG), Madison, Wis.) was used to align the human genomic sequencesurrounding the EST-S4 with the rat 90 kDa DAGKβ mRNA. The EST-S4 islocated in the 30 Kb intron between exons b and c. It overlaps with theAC005039 genomic sequence 91 bp downstream the last amino acid of exonb. The genomic sequence contains two in frame stop codons and a possiblepolyadenylation signal is present on the EST sequence.

In order to verify if EST-S4 identifies a splice variant of the humanDAGKβ gene, a reverse transcriptase polymerase chain reaction (RT-PCR)using the following primer pair: DAGK7for (5′TGCCMTGCAAATTGATGGG) andDAGK7rev (5′AGCTAAATCATTGCCAAGGG) that span exon b and EST-S4 wasperformed. The PCR conditions included an initial hot-start at 94° C.for 5 minutes, followed by 35 cycles at 94° C. for 1 minute, 56° C. for1 minute and 72° C. for 1 minute and terminated by 5 minutes at 72° C.The resulting PCR amplicon was separated on a 2% agarose gel and shownto be of 246 bp.

The transcript was successfully amplified in both human adult and foetalbrain polyA+RNA (Clontech) RT-PCR reactions. As a control the humangenomic DNA was also amplified, yielding a fragment of the same length.The results indicate that the EST-S4 might correspond to the 3′-UTR of anew splice variant (FIG. 1, panel A). Thus the shortest form of hDAGKβprotein encoded by the splice variant transcripts is herein designatedas SV-hDAGKβ.

In order to further confirm the existence of an alternative splicingevent giving rise to a 3′ splice variant hDAGKβ isoform a 3′ rapidamplification of cDNA ends (RACE) strategy (19) was applied tophysically identify and clone the relevant transcript portion in theform of cDNA. Briefly, human foetal brain polyadenylated RNA was reversetranscribed using the anchor oligonucleotideCCAGTGAGCAGAGTGACGAGGACTCGAGCTCAAGC(T)₁₇ as a primer for first strandcDNA synthesis. The resulting cDNA was employed as a template for twonested rounds of PCR employing anchor-specific and gene-specific primers(first round RACE: CCAGTGAGCAGAGTGACG and TCAGAGCCACTACATTTAGGT; secondround RACE: GAGGACTCGAGCTCAAGC and AGGTTGTAGACATTATATACC). PCRconditions for both rounds were: 94° C. for 3 minutes (hot start); 25cycles of (94° C. for 30″, 56° C. for 30″, 72° C. for 30″) followed by a72° C. for 10′ step. Two RACE products, of 200 bp and 750 bprespectively, were obtained and cloned into appropriate E. coli plasmidcloning vectors. Double-pass sequencing confirmed the identity of thetwo products as two alternatively spliced transcripts bearing the 3′ endof the human DAGKβ splice variant coding sequence (with two predictedin-frame translational stop codons at position 2320 and 2365) and ca.100 bp or ca. 650 bp (owing to two alternatively used polyadenylationsignals) of 3′ untranslated sequence (UTR). Thus hDAGKβ splice varianttranscripts run out of the penultimate coding exon into the last intronof the DAGKβ locus, terminating at two alternatively usedpolyadenylation signals. The predicted protein encoded by the splicevariant transcripts (Seq ID No 4) lacks the last 30 amino acids presentin the longest (direct human orthologue of the rat DAGKβ) variant.

Physical Cloning of the Full Length Sequence of Human hDAGKβ andSV-hDAGKβ cDNAs

In order to clone the full length cDNA sequence encoding the two DAGKβvariants, the sequence information derived from the in silico andcloning analyses was employed to design PCR primers comprising theinitiation codon of the protein (common to both variants) and thesequence immediately 3′ of the predicted translational stop codons ofeach variant. These primers were employed in two successive rounds ofnested long-range PCR (LR-PCR) employing a proofreading thermostablepolymerase (XL-PCR kit, Perkin Elmer, Calif.) according tomanufacturer's instructions. First round PCR conditions were as follows:94° C. for 3 minutes (hot start); 35 cycles of (94° C. for 30″, 55° C.for 30″, 72° C. for 5′) followed by a 72° C. for 20′ step. Second roundPCR was carried out on a 1 μl aliquot of a 1:10 dilution of first roundPCR using the same conditions as for first round PCR, except thatcycling was for 25 cycles. Primers were as detailed in Table 1. Theresulting PCR-amplified products (ca. 2.4 kb) were cloned intoappropriate plasmid cloning vectors and subjected to double passsequence analysis. This exercise confirmed the cDNA sequence predictedby the in silico analysis and extended it by revealing the presence ofthree alternatively spliced exons within hDAGKβ and SV-hDAGKβtranscripts and derived cDNAs (FIG. 5).

Alternative Transcript Splicing Generates Several Isoforms of the hDAGKβand SV-hDAGKβ

Sequence analysis of the cloned full length cDNAs revealed the presenceof three alternatively spliced exons (encoding amino acid sequences of7, 12 and 25 residues respectively) in addition to the previouslycharacterized alternative splicing event leading to the generation ofSV-hDAGKβ transcripts (FIG. 2). Thus at least 8 hDAGKβ isoforms and 8SV-hDAGKβ isoforms are predicted.

Expression of hDAGKβ and SV-hDAGKβ Transcripts in Adult Human Tissues

The specificity of hDAGKβ and SV-hDAGKβ expression in adult humantissues was investigated by RT-PCR. Polyadenylated RNA for a variety ofhuman tissues was obtained from a commercial source (Clontech, Calif.).This material was converted to cDNA and a set of PCR primers capable ofselectively amplifying either the hDAGKβ or the SV isoform were employedin an RT-PCR study. A set of β-actin specific primers were employed tocontrol the efficacy of the RT-PCR process. Table 1 provides the detailsof the primers. PCR conditions were: 94° C. for 3 minutes (hot start);40 cycles of (94° C. for 30″, 56° C. for 30″, 72° C. for 30″) followedby a 72° C. for 10′ step. The results (FIG. 3) indicate that both hDAGKβand SV-hDAGKβ are coordinately expressed in all tissues of neuronalorigin (brain regions and spinal cord). Non-neuronal tissues (with theexception of the uterus) do not express significant levels of hDAGKβtranscripts.

TABLE 1 PCR primers used in the present study. A. PCR primers employedfor the amplification of full length hDAGKβ cDNAs. The initiation(start) codon is underlined. B. PCR primers for the analysis ofexpression of h DAGKβ and SV-hDAGKβ isoforms. Sizes of expected productsare indicated. A DAGKβ transcript First round Second round hDAGKβCACCACCATGACAAACCAGG ATGACAAACCAGGAAAAATGG and and TCTAAGAGTGAAACAACACACAGGATTATTCCTTGCTTCGG SV-hDAGKβ CACCACCATGACAAACCAGGATGACAAACCAGGAAAAATGG and and AGCTAAATCATTGCCAAGG TCTACAACCTAAATGTAGTGGB DAGKβ transcript Primers amplified product (bp) hDAKGβTGCCAATGCAAATTGA 153 and AGGATTATTCCTTGCTTCGG SV-hDAGKβ TGCCAATGCAAATTGA246 and AGCTAAATCATTGCCAAG β-actin TGAACCCTAAGGCCAACCGTG 400 andGCTCATAGCTCTTCTCCAGGG

Expression of hDAGKβ Transcripts in Normal Vs NeuropathologicalConditions

The association between the hDAGKβ and the disorders arising fromabnormal expression/activity of hDAGKβ protein and variants thereof canbe illustrated by the following experiments.

The expression of hDAGKβ transcripts in normal and pathologicalconditions of relevance to the present patent was initiated by analyzinghDAGKβ expression by RT-PCR in cDNA from the cerebellum of normalindividuals and Alzheimer cerebellum. An initial analysis was carriedout using a set of hDAGKβ specific and SV-hDAGKβ specific primers andPCR conditions as detailed in the above section. The results (FIG. 4)indicate that expression of hDAGKβ is lost in Alzheimer cerebellum,while expression of SV-hDAGKβ is unchanged.

Screening for Compounds which Exhibit hDAGKβ Modulating Activity

To identify modulators of hDAGK activity (especially hDAGKβ) activitylike inhibitors and activators, respectively, a cellular homogenatecontaining the hDAGK polypeptide (either from cells transfected with theDAGK cDNA or from overproducing organisms) is incubated with substrates(like DAG and ATP) in the absence or the presence of a chemical entityor crude natural extract that might modulate hDAGK activity (primaryscreening). The activity of the hDAGK polypeptide or a purifiedpreparation of hDAGK polypeptide in the reaction mixture can bequantified by measuring the ATP-dependent phosphorylation of the DAGsubstrate employing radio-labelled ATP as substrate (20). The labelledproduct (phosphatidic acid) is extracted into acidified organic solventsand quantitated by scintillation counting. For more accuratedeterminations the phosphatidic acid product can also be separated fromthe mixture by TLC methods and the corresponding radioactive bands canbe quantified by using a phosphoimager system. Fluorescence orchemiluminescence-tagged DAG can also be used as the substrate forhDAGK. In this case, the PA product will become labelled with theselected probe and can be separated from the substrate donor molecule byTLC and quantified via densitometric analysis of fluorescent orchemoluminescent spots.

This application also relates to a method of identifying a compound or acomposition that can activate or inhibit the activity of the promoterfor the DNA of the present invention, which comprises (i) adding a testcompound to a cell line whose hDAGK gene has been inactivated byintroducing a reporter gene, e.g., the beta-galactosidase from E. coliorigin (lacZ); (ii) determining if transcription of the reporter geneoccurs by measuring the level of activation of lacZ gene using thechromogenic substrate X-gal using high-throughput calorimetricmeasurements. The compounds or compositions which are able to inhibit oractivate the promoter for the hDAGKβ DNA will alter the expression ofthe reporter gene. Compounds identified in this way are then tested invivo to assess their ability to modulate the level of the expression ofhDAGK, especially hDAGKβ, in mice CNS.

It is to be understood that modifications and/or alterations to theaspects of the invention specifically disclosed within this application,which based upon the disclosure herein would be readily apparent to aperson skilled in the art, are also considered to be included within thescope of the invention as outlined in the appended claims.

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7 1 3926 DNA Homo sapiens CDS (1)..(2415) 1 atg aca aac cag gaa aaa tgggcc cac ctc agc cct tcg gaa ttt tcc 48 Met Thr Asn Gln Glu Lys Trp AlaHis Leu Ser Pro Ser Glu Phe Ser 1 5 10 15 caa ctt cag aaa tat gct gagtat tct aca aag aaa tta aag gat gtt 96 Gln Leu Gln Lys Tyr Ala Glu TyrSer Thr Lys Lys Leu Lys Asp Val 20 25 30 ctt gaa gaa ttc cat ggt aat ggtgtg ctt gca aag tat aat cct gaa 144 Leu Glu Glu Phe His Gly Asn Gly ValLeu Ala Lys Tyr Asn Pro Glu 35 40 45 ggg aaa caa gac att ctt aac caa acaata gat ttt gaa ggt ttc aaa 192 Gly Lys Gln Asp Ile Leu Asn Gln Thr IleAsp Phe Glu Gly Phe Lys 50 55 60 cta ttc atg aag aca ttc ctg gaa gcc gagctt cct gat gat ttc act 240 Leu Phe Met Lys Thr Phe Leu Glu Ala Glu LeuPro Asp Asp Phe Thr 65 70 75 80 gca cac ctt ttc atg tca ttt agc aac aagttt cct cat tct agt cca 288 Ala His Leu Phe Met Ser Phe Ser Asn Lys PhePro His Ser Ser Pro 85 90 95 atg gta aaa agt aag cct gct ctc cta tca ggcggt ctg aga atg aat 336 Met Val Lys Ser Lys Pro Ala Leu Leu Ser Gly GlyLeu Arg Met Asn 100 105 110 aaa ggt gcc atc acc cct ccc cga act act tctcct gca aat acg tgt 384 Lys Gly Ala Ile Thr Pro Pro Arg Thr Thr Ser ProAla Asn Thr Cys 115 120 125 tcc cca gaa gta atc cat ctg aag gac att gtctgt tac ctg tct ctg 432 Ser Pro Glu Val Ile His Leu Lys Asp Ile Val CysTyr Leu Ser Leu 130 135 140 ctt gaa aga gga aga cct gag gat aag ctt gagttt atg ttt cgc ctt 480 Leu Glu Arg Gly Arg Pro Glu Asp Lys Leu Glu PheMet Phe Arg Leu 145 150 155 160 tat gac acg gat ggg aat ggc ttc ctg gacagc tcg gag cta gaa aat 528 Tyr Asp Thr Asp Gly Asn Gly Phe Leu Asp SerSer Glu Leu Glu Asn 165 170 175 atc atc agt cag atg atg cat gtt gca gaatac ctt gag tgg gat gtc 576 Ile Ile Ser Gln Met Met His Val Ala Glu TyrLeu Glu Trp Asp Val 180 185 190 act gaa ctt aat cca atc ctc cat gaa atgatg gaa gaa att gac tat 624 Thr Glu Leu Asn Pro Ile Leu His Glu Met MetGlu Glu Ile Asp Tyr 195 200 205 gat cat gat gga acc gtg tct ctg gag gaatgg att caa gga gga atg 672 Asp His Asp Gly Thr Val Ser Leu Glu Glu TrpIle Gln Gly Gly Met 210 215 220 aca acg att cca ctt ctt gtg ctc ctg ggctta gaa aat aac gtg aag 720 Thr Thr Ile Pro Leu Leu Val Leu Leu Gly LeuGlu Asn Asn Val Lys 225 230 235 240 gat gat gga cag cac gtg tgg cga ctgaag cac ttt aac aaa cct gcc 768 Asp Asp Gly Gln His Val Trp Arg Leu LysHis Phe Asn Lys Pro Ala 245 250 255 tat tgc aac ctt tgc ctg aac atg ctgatt ggc gtg ggg aag cag ggc 816 Tyr Cys Asn Leu Cys Leu Asn Met Leu IleGly Val Gly Lys Gln Gly 260 265 270 ctc tgc tgt tcc ttc tgc aag tac acagtc cat gag cgc tgt gtg gct 864 Leu Cys Cys Ser Phe Cys Lys Tyr Thr ValHis Glu Arg Cys Val Ala 275 280 285 cga gca cct ccc tct tgc atc aag acctat gtg aag tcc aaa agg aac 912 Arg Ala Pro Pro Ser Cys Ile Lys Thr TyrVal Lys Ser Lys Arg Asn 290 295 300 act gat gtc atg cac cat tac tgg gttgaa ggt aac tgc cca acc aag 960 Thr Asp Val Met His His Tyr Trp Val GluGly Asn Cys Pro Thr Lys 305 310 315 320 tgt gat aag tgc cac aaa act gttaaa tgt tac cag ggc ctg aca gga 1008 Cys Asp Lys Cys His Lys Thr Val LysCys Tyr Gln Gly Leu Thr Gly 325 330 335 ctg cat tgt gtt tgg tgt cag atcaca ctg cat aat aaa tgt gct tct 1056 Leu His Cys Val Trp Cys Gln Ile ThrLeu His Asn Lys Cys Ala Ser 340 345 350 cat cta aaa cct gaa tgt gac tgtgga cct ttg aag gac cat att tta 1104 His Leu Lys Pro Glu Cys Asp Cys GlyPro Leu Lys Asp His Ile Leu 355 360 365 cca ccc aca aca atc tgt cca gtggta ctg cag act ctg ccc act tca 1152 Pro Pro Thr Thr Ile Cys Pro Val ValLeu Gln Thr Leu Pro Thr Ser 370 375 380 gga gtt tca gtt cct gag gaa agacaa tca aca gtg aaa aag gaa aag 1200 Gly Val Ser Val Pro Glu Glu Arg GlnSer Thr Val Lys Lys Glu Lys 385 390 395 400 agt ggt tcc cag cag cca aacaaa gtg att gac aag aat aaa atg caa 1248 Ser Gly Ser Gln Gln Pro Asn LysVal Ile Asp Lys Asn Lys Met Gln 405 410 415 aga gcc aac tct gtt act gtagat gga caa ggc ctg cag gtc act cct 1296 Arg Ala Asn Ser Val Thr Val AspGly Gln Gly Leu Gln Val Thr Pro 420 425 430 gtg cct ggt act cac cca ctttta gtt ttt gtg aac ccc aaa agt ggt 1344 Val Pro Gly Thr His Pro Leu LeuVal Phe Val Asn Pro Lys Ser Gly 435 440 445 gga aaa caa gga gaa cga atttac aga aaa ttc cag tat cta tta aat 1392 Gly Lys Gln Gly Glu Arg Ile TyrArg Lys Phe Gln Tyr Leu Leu Asn 450 455 460 cct cgt cag gtt tac agt ctttct gga aat gga cca atg cca ggg tta 1440 Pro Arg Gln Val Tyr Ser Leu SerGly Asn Gly Pro Met Pro Gly Leu 465 470 475 480 aac ttt ttc cgt gat gttcct gac ttc aga gtg tta gcc tgt ggt gga 1488 Asn Phe Phe Arg Asp Val ProAsp Phe Arg Val Leu Ala Cys Gly Gly 485 490 495 gat gga acc gtg ggc tgggtt ttg gat tgc ata gaa aag gcc aat gta 1536 Asp Gly Thr Val Gly Trp ValLeu Asp Cys Ile Glu Lys Ala Asn Val 500 505 510 ggc aag cat cct cca gttgcg att ctg cct ctt ggg act ggc aat gat 1584 Gly Lys His Pro Pro Val AlaIle Leu Pro Leu Gly Thr Gly Asn Asp 515 520 525 cta gca aga tgc ctg cgatgg gga gga ggt tac gaa ggt gag aat ctg 1632 Leu Ala Arg Cys Leu Arg TrpGly Gly Gly Tyr Glu Gly Glu Asn Leu 530 535 540 atg aaa att cta aaa gacatt gaa aac agc aca gaa atc atg ttg gac 1680 Met Lys Ile Leu Lys Asp IleGlu Asn Ser Thr Glu Ile Met Leu Asp 545 550 555 560 agg tgg aag ttt gaagtc ata cct aat gac aaa gat gag aaa gga gac 1728 Arg Trp Lys Phe Glu ValIle Pro Asn Asp Lys Asp Glu Lys Gly Asp 565 570 575 cca gtg cct tac agtatc atc aat aat tac ttt tcc att ggc gtg gat 1776 Pro Val Pro Tyr Ser IleIle Asn Asn Tyr Phe Ser Ile Gly Val Asp 580 585 590 gcc tcc att gca cacaga ttc cac atc atg aga gaa aaa cac cca gag 1824 Ala Ser Ile Ala His ArgPhe His Ile Met Arg Glu Lys His Pro Glu 595 600 605 aaa ttc aac agt agaatg aag aac aaa ttt tgg tat ttt gag ttt ggc 1872 Lys Phe Asn Ser Arg MetLys Asn Lys Phe Trp Tyr Phe Glu Phe Gly 610 615 620 aca tct gaa act ttctca gcc acc tgc aag aag cta cat gaa tct gta 1920 Thr Ser Glu Thr Phe SerAla Thr Cys Lys Lys Leu His Glu Ser Val 625 630 635 640 gaa ata gaa tgtgat gga gta cag ata gat tta ata aac atc tct ctg 1968 Glu Ile Glu Cys AspGly Val Gln Ile Asp Leu Ile Asn Ile Ser Leu 645 650 655 gaa gga att gctatt ttg aat ata cca agc atg cat gga gga tcc aat 2016 Glu Gly Ile Ala IleLeu Asn Ile Pro Ser Met His Gly Gly Ser Asn 660 665 670 ctt tgg gga gagtct aag aaa aga cga agc cat cga cga ata gag aaa 2064 Leu Trp Gly Glu SerLys Lys Arg Arg Ser His Arg Arg Ile Glu Lys 675 680 685 aaa ggg tct gacaaa agg acc acc gtc aca gat gcc aaa gag ttg aag 2112 Lys Gly Ser Asp LysArg Thr Thr Val Thr Asp Ala Lys Glu Leu Lys 690 695 700 ttt gca agt caagat ctc agt gac cag ctg ctg gag gtg gtc ggc ttg 2160 Phe Ala Ser Gln AspLeu Ser Asp Gln Leu Leu Glu Val Val Gly Leu 705 710 715 720 gaa gga gccatg gag atg ggg caa ata tac aca ggc ctg aaa agt gct 2208 Glu Gly Ala MetGlu Met Gly Gln Ile Tyr Thr Gly Leu Lys Ser Ala 725 730 735 ggc cgg cggctg gct cag tgc tcc tgc gtg gtc atc agg acg agc aag 2256 Gly Arg Arg LeuAla Gln Cys Ser Cys Val Val Ile Arg Thr Ser Lys 740 745 750 tct ctg ccaatg caa att gat ggg gag cca tgg atg cag acc cca tgc 2304 Ser Leu Pro MetGln Ile Asp Gly Glu Pro Trp Met Gln Thr Pro Cys 755 760 765 aca ata aaaatt aca cac aag aac caa gcc cca atg ctg atg ggc ccg 2352 Thr Ile Lys IleThr His Lys Asn Gln Ala Pro Met Leu Met Gly Pro 770 775 780 cct cca aaaacc ggt tta ttc tgc tcc ctc gtc aaa agg aca aga aac 2400 Pro Pro Lys ThrGly Leu Phe Cys Ser Leu Val Lys Arg Thr Arg Asn 785 790 795 800 cga agcaag gaa taa tcctgtgttg tttcactctt agaaattgaa ttagcataat 2455 Arg Ser LysGlu tgggccatgg aacacatatg ctggaaatct ttgaaccatt tcaagtctcc tgctcatgca2515 aaatcatgga agtggtttaa cagtttttgt tactaagcta atgtaaaatt cagctattag2575 aaaatttatt gtctcagttt ttataggcat ctttgcatga agaaagcaga agtttacctg2635 aagtgatact gcatattttt ggtgcatgca ttcccataga tttttacatc tcccacccaa2695 ctcttcccca atttcctttt actaacctgt gagaaaaacc cgtgaaacat gaaaaaggaa2755 ataccatggg aaacgtgatt ctcagtgtga ttccaattat tacgaagcac taatcagtaa2815 cgctacaatg atcataattg cagattgcta tacgtttccc ttttagaatc agtgtatcag2875 tgacctatga cttgaggaga aacttttaat tcgaagattt tattaaatag ttgactacaa2935 taccttgcta tatatacata gtttttcttc aacatcttaa ctcttctgag tggaaataaa2995 aatatcaggc ataaggtttt ctcatgctga aaaatagaac gcggttttta ttttgcttag3055 ttttcttttt aattccagaa ataagtgaaa acatgttact tgacagtcaa gtgtggtaat3115 atggcaagcc ttgttccttt ctgcatgaga atctaggaga gaattcataa ccacaccaat3175 aacgaaatag aagttttaaa ctatgtgcct aatcaatgtg tttcccacca aagattcaga3235 aaacaatgct tgagagaaat gggttaatgc ataattaatt aagcattgtg gagcaaattt3295 agggttcctg tgattaattt tgtgatgact aaaatgctgg aaagcaagtg agttgcccat3355 taattatgat taaaattctc acctttcaca gacagacaat aagccagaca acacaatcaa3415 agctcaatag atgatttctt gcttttttca gtcatttata aatataggtg taatttttca3475 tggatcagtt aagtacactt gaaggaagta aatgattgta tcagtttatt tctagtataa3535 atgggtacct gtaataatac tgagctcttg gaagcgaatc atgcatgcaa ttagctccct3595 cctcctcacc tactccactc ccatctttat gacatttcaa atgtttattt ggaaacaaca3655 gcctagatca ctgttgaagg tgttcatggc atagttggag tctctgactg tttaaagaaa3715 tcacagaaca gtacttttct tttagtgttt cattaagcct atgatgtaaa atgaaatgct3775 tctgagcagt cttgtaatat tgttcattca tattgacctg catctcatca ttgcatgttt3835 tatgttttca aacatgccat aaggaaaacg agtgcctgaa ctgcatgatt tattagtttc3895 tctccactct gcattaaagt gctaatgatt t 3926 2 804 PRT Homo sapiens 2Met Thr Asn Gln Glu Lys Trp Ala His Leu Ser Pro Ser Glu Phe Ser 1 5 1015 Gln Leu Gln Lys Tyr Ala Glu Tyr Ser Thr Lys Lys Leu Lys Asp Val 20 2530 Leu Glu Glu Phe His Gly Asn Gly Val Leu Ala Lys Tyr Asn Pro Glu 35 4045 Gly Lys Gln Asp Ile Leu Asn Gln Thr Ile Asp Phe Glu Gly Phe Lys 50 5560 Leu Phe Met Lys Thr Phe Leu Glu Ala Glu Leu Pro Asp Asp Phe Thr 65 7075 80 Ala His Leu Phe Met Ser Phe Ser Asn Lys Phe Pro His Ser Ser Pro 8590 95 Met Val Lys Ser Lys Pro Ala Leu Leu Ser Gly Gly Leu Arg Met Asn100 105 110 Lys Gly Ala Ile Thr Pro Pro Arg Thr Thr Ser Pro Ala Asn ThrCys 115 120 125 Ser Pro Glu Val Ile His Leu Lys Asp Ile Val Cys Tyr LeuSer Leu 130 135 140 Leu Glu Arg Gly Arg Pro Glu Asp Lys Leu Glu Phe MetPhe Arg Leu 145 150 155 160 Tyr Asp Thr Asp Gly Asn Gly Phe Leu Asp SerSer Glu Leu Glu Asn 165 170 175 Ile Ile Ser Gln Met Met His Val Ala GluTyr Leu Glu Trp Asp Val 180 185 190 Thr Glu Leu Asn Pro Ile Leu His GluMet Met Glu Glu Ile Asp Tyr 195 200 205 Asp His Asp Gly Thr Val Ser LeuGlu Glu Trp Ile Gln Gly Gly Met 210 215 220 Thr Thr Ile Pro Leu Leu ValLeu Leu Gly Leu Glu Asn Asn Val Lys 225 230 235 240 Asp Asp Gly Gln HisVal Trp Arg Leu Lys His Phe Asn Lys Pro Ala 245 250 255 Tyr Cys Asn LeuCys Leu Asn Met Leu Ile Gly Val Gly Lys Gln Gly 260 265 270 Leu Cys CysSer Phe Cys Lys Tyr Thr Val His Glu Arg Cys Val Ala 275 280 285 Arg AlaPro Pro Ser Cys Ile Lys Thr Tyr Val Lys Ser Lys Arg Asn 290 295 300 ThrAsp Val Met His His Tyr Trp Val Glu Gly Asn Cys Pro Thr Lys 305 310 315320 Cys Asp Lys Cys His Lys Thr Val Lys Cys Tyr Gln Gly Leu Thr Gly 325330 335 Leu His Cys Val Trp Cys Gln Ile Thr Leu His Asn Lys Cys Ala Ser340 345 350 His Leu Lys Pro Glu Cys Asp Cys Gly Pro Leu Lys Asp His IleLeu 355 360 365 Pro Pro Thr Thr Ile Cys Pro Val Val Leu Gln Thr Leu ProThr Ser 370 375 380 Gly Val Ser Val Pro Glu Glu Arg Gln Ser Thr Val LysLys Glu Lys 385 390 395 400 Ser Gly Ser Gln Gln Pro Asn Lys Val Ile AspLys Asn Lys Met Gln 405 410 415 Arg Ala Asn Ser Val Thr Val Asp Gly GlnGly Leu Gln Val Thr Pro 420 425 430 Val Pro Gly Thr His Pro Leu Leu ValPhe Val Asn Pro Lys Ser Gly 435 440 445 Gly Lys Gln Gly Glu Arg Ile TyrArg Lys Phe Gln Tyr Leu Leu Asn 450 455 460 Pro Arg Gln Val Tyr Ser LeuSer Gly Asn Gly Pro Met Pro Gly Leu 465 470 475 480 Asn Phe Phe Arg AspVal Pro Asp Phe Arg Val Leu Ala Cys Gly Gly 485 490 495 Asp Gly Thr ValGly Trp Val Leu Asp Cys Ile Glu Lys Ala Asn Val 500 505 510 Gly Lys HisPro Pro Val Ala Ile Leu Pro Leu Gly Thr Gly Asn Asp 515 520 525 Leu AlaArg Cys Leu Arg Trp Gly Gly Gly Tyr Glu Gly Glu Asn Leu 530 535 540 MetLys Ile Leu Lys Asp Ile Glu Asn Ser Thr Glu Ile Met Leu Asp 545 550 555560 Arg Trp Lys Phe Glu Val Ile Pro Asn Asp Lys Asp Glu Lys Gly Asp 565570 575 Pro Val Pro Tyr Ser Ile Ile Asn Asn Tyr Phe Ser Ile Gly Val Asp580 585 590 Ala Ser Ile Ala His Arg Phe His Ile Met Arg Glu Lys His ProGlu 595 600 605 Lys Phe Asn Ser Arg Met Lys Asn Lys Phe Trp Tyr Phe GluPhe Gly 610 615 620 Thr Ser Glu Thr Phe Ser Ala Thr Cys Lys Lys Leu HisGlu Ser Val 625 630 635 640 Glu Ile Glu Cys Asp Gly Val Gln Ile Asp LeuIle Asn Ile Ser Leu 645 650 655 Glu Gly Ile Ala Ile Leu Asn Ile Pro SerMet His Gly Gly Ser Asn 660 665 670 Leu Trp Gly Glu Ser Lys Lys Arg ArgSer His Arg Arg Ile Glu Lys 675 680 685 Lys Gly Ser Asp Lys Arg Thr ThrVal Thr Asp Ala Lys Glu Leu Lys 690 695 700 Phe Ala Ser Gln Asp Leu SerAsp Gln Leu Leu Glu Val Val Gly Leu 705 710 715 720 Glu Gly Ala Met GluMet Gly Gln Ile Tyr Thr Gly Leu Lys Ser Ala 725 730 735 Gly Arg Arg LeuAla Gln Cys Ser Cys Val Val Ile Arg Thr Ser Lys 740 745 750 Ser Leu ProMet Gln Ile Asp Gly Glu Pro Trp Met Gln Thr Pro Cys 755 760 765 Thr IleLys Ile Thr His Lys Asn Gln Ala Pro Met Leu Met Gly Pro 770 775 780 ProPro Lys Thr Gly Leu Phe Cys Ser Leu Val Lys Arg Thr Arg Asn 785 790 795800 Arg Ser Lys Glu 3 3926 DNA Homo sapiens 3 aaatcattag cactttaatgcagagtggag agaaactaat aaatcatgca gttcaggcac 60 tcgttttcct tatggcatgtttgaaaacat aaaacatgca atgatgagat gcaggtcaat 120 atgaatgaac aatattacaagactgctcag aagcatttca ttttacatca taggcttaat 180 gaaacactaa aagaaaagtactgttctgtg atttctttaa acagtcagag actccaacta 240 tgccatgaac accttcaacagtgatctagg ctgttgtttc caaataaaca tttgaaatgt 300 cataaagatg ggagtggagtaggtgaggag gagggagcta attgcatgca tgattcgctt 360 ccaagagctc agtattattacaggtaccca tttatactag aaataaactg atacaatcat 420 ttacttcctt caagtgtacttaactgatcc atgaaaaatt acacctatat ttataaatga 480 ctgaaaaaag caagaaatcatctattgagc tttgattgtg ttgtctggct tattgtctgt 540 ctgtgaaagg tgagaattttaatcataatt aatgggcaac tcacttgctt tccagcattt 600 tagtcatcac aaaattaatcacaggaaccc taaatttgct ccacaatgct taattaatta 660 tgcattaacc catttctctcaagcattgtt ttctgaatct ttggtgggaa acacattgat 720 taggcacata gtttaaaacttctatttcgt tattggtgtg gttatgaatt ctctcctaga 780 ttctcatgca gaaaggaacaaggcttgcca tattaccaca cttgactgtc aagtaacatg 840 ttttcactta tttctggaattaaaaagaaa actaagcaaa ataaaaaccg cgttctattt 900 ttcagcatga gaaaaccttatgcctgatat ttttatttcc actcagaaga gttaagatgt 960 tgaagaaaaa ctatgtatatatagcaaggt attgtagtca actatttaat aaaatcttcg 1020 aattaaaagt ttctcctcaagtcataggtc actgatacac tgattctaaa agggaaacgt 1080 atagcaatct gcaattatgatcattgtagc gttactgatt agtgcttcgt aataattgga 1140 atcacactga gaatcacgtttcccatggta tttccttttt catgtttcac gggtttttct 1200 cacaggttag taaaaggaaattggggaaga gttgggtggg agatgtaaaa atctatggga 1260 atgcatgcac caaaaatatgcagtatcact tcaggtaaac ttctgctttc ttcatgcaaa 1320 gatgcctata aaaactgagacaataaattt tctaatagct gaattttaca ttagcttagt 1380 aacaaaaact gttaaaccacttccatgatt ttgcatgagc aggagacttg aaatggttca 1440 aagatttcca gcatatgtgttccatggccc aattatgcta attcaatttc taagagtgaa 1500 acaacacagg attattccttgcttcggttt cttgtccttt tgacgaggga gcagaataaa 1560 ccggtttttg gaggcgggcccatcagcatt ggggcttggt tcttgtgtgt aatttttatt 1620 gtgcatgggg tctgcatccatggctcccca tcaatttgca ttggcagaga cttgctcgtc 1680 ctgatgacca cgcaggagcactgagccagc cgccggccag cacttttcag gcctgtgtat 1740 atttgcccca tctccatggctccttccaag ccgaccacct ccagcagctg gtcactgaga 1800 tcttgacttg caaacttcaactctttggca tctgtgacgg tggtcctttt gtcagaccct 1860 tttttctcta ttcgtcgatggcttcgtctt ttcttagact ctccccaaag attggatcct 1920 ccatgcatgc ttggtatattcaaaatagca attccttcca gagagatgtt tattaaatct 1980 atctgtactc catcacattctatttctaca gattcatgta gcttcttgca ggtggctgag 2040 aaagtttcag atgtgccaaactcaaaatac caaaatttgt tcttcattct actgttgaat 2100 ttctctgggt gtttttctctcatgatgtgg aatctgtgtg caatggaggc atccacgcca 2160 atggaaaagt aattattgatgatactgtaa ggcactgggt ctcctttctc atctttgtca 2220 ttaggtatga cttcaaacttccacctgtcc aacatgattt ctgtgctgtt ttcaatgtct 2280 tttagaattt tcatcagattctcaccttcg taacctcctc cccatcgcag gcatcttgct 2340 agatcattgc cagtcccaagaggcagaatc gcaactggag gatgcttgcc tacattggcc 2400 ttttctatgc aatccaaaacccagcccacg gttccatctc caccacaggc taacactctg 2460 aagtcaggaa catcacggaaaaagtttaac cctggcattg gtccatttcc agaaagactg 2520 taaacctgac gaggatttaatagatactgg aattttctgt aaattcgttc tccttgtttt 2580 ccaccacttt tggggttcacaaaaactaaa agtgggtgag taccaggcac aggagtgacc 2640 tgcaggcctt gtccatctacagtaacagag ttggctcttt gcattttatt cttgtcaatc 2700 actttgtttg gctgctgggaaccactcttt tcctttttca ctgttgattg tctttcctca 2760 ggaactgaaa ctcctgaagtgggcagagtc tgcagtacca ctggacagat tgttgtgggt 2820 ggtaaaatat ggtccttcaaaggtccacag tcacattcag gttttagatg agaagcacat 2880 ttattatgca gtgtgatctgacaccaaaca caatgcagtc ctgtcaggcc ctggtaacat 2940 ttaacagttt tgtggcacttatcacacttg gttgggcagt taccttcaac ccagtaatgg 3000 tgcatgacat cagtgttccttttggacttc acataggtct tgatgcaaga gggaggtgct 3060 cgagccacac agcgctcatggactgtgtac ttgcagaagg aacagcagag gccctgcttc 3120 cccacgccaa tcagcatgttcaggcaaagg ttgcaatagg caggtttgtt aaagtgcttc 3180 agtcgccaca cgtgctgtccatcatccttc acgttatttt ctaagcccag gagcacaaga 3240 agtggaatcg ttgtcattcctccttgaatc cattcctcca gagacacggt tccatcatga 3300 tcatagtcaa tttcttccatcatttcatgg aggattggat taagttcagt gacatcccac 3360 tcaaggtatt ctgcaacatgcatcatctga ctgatgatat tttctagctc cgagctgtcc 3420 aggaagccat tcccatccgtgtcataaagg cgaaacataa actcaagctt atcctcaggt 3480 cttcctcttt caagcagagacaggtaacag acaatgtcct tcagatggat tacttctggg 3540 gaacacgtat ttgcaggagaagtagttcgg ggaggggtga tggcaccttt attcattctc 3600 agaccgcctg ataggagagcaggcttactt tttaccattg gactagaatg aggaaacttg 3660 ttgctaaatg acatgaaaaggtgtgcagtg aaatcatcag gaagctcggc ttccaggaat 3720 gtcttcatga atagtttgaaaccttcaaaa tctattgttt ggttaagaat gtcttgtttc 3780 ccttcaggat tatactttgcaagcacacca ttaccatgga attcttcaag aacatccttt 3840 aatttctttg tagaatactcagcatatttc tgaagttggg aaaattccga agggctgagg 3900 tgggcccatt tttcctggtttgtcat 3926 4 3172 DNA Homo sapiens CDS (1)..(2322) 4 atg aca aac caggaa aaa tgg gcc cac ctc agc cct tcg gaa ttt tcc 48 Met Thr Asn Gln GluLys Trp Ala His Leu Ser Pro Ser Glu Phe Ser 1 5 10 15 caa ctt cag aaatat gct gag tat tct aca aag aaa tta aag gat gtt 96 Gln Leu Gln Lys TyrAla Glu Tyr Ser Thr Lys Lys Leu Lys Asp Val 20 25 30 ctt gaa gaa ttc catggt aat ggt gtg ctt gca aag tat aat cct gaa 144 Leu Glu Glu Phe His GlyAsn Gly Val Leu Ala Lys Tyr Asn Pro Glu 35 40 45 ggg aaa caa gac att cttaac caa aca ata gat ttt gaa ggt ttc aaa 192 Gly Lys Gln Asp Ile Leu AsnGln Thr Ile Asp Phe Glu Gly Phe Lys 50 55 60 cta ttc atg aag aca ttc ctggaa gcc gag ctt cct gat gat ttc act 240 Leu Phe Met Lys Thr Phe Leu GluAla Glu Leu Pro Asp Asp Phe Thr 65 70 75 80 gca cac ctt ttc atg tca tttagc aac aag ttt cct cat tct agt cca 288 Ala His Leu Phe Met Ser Phe SerAsn Lys Phe Pro His Ser Ser Pro 85 90 95 atg gta aaa agt aag cct gct ctccta tca ggc ggt ctg aga atg aat 336 Met Val Lys Ser Lys Pro Ala Leu LeuSer Gly Gly Leu Arg Met Asn 100 105 110 aaa ggt gcc atc acc cct ccc cgaact act tct cct gca aat acg tgt 384 Lys Gly Ala Ile Thr Pro Pro Arg ThrThr Ser Pro Ala Asn Thr Cys 115 120 125 tcc cca gaa gta atc cat ctg aaggac att gtc tgt tac ctg tct ctg 432 Ser Pro Glu Val Ile His Leu Lys AspIle Val Cys Tyr Leu Ser Leu 130 135 140 ctt gaa aga gga aga cct gag gataag ctt gag ttt atg ttt cgc ctt 480 Leu Glu Arg Gly Arg Pro Glu Asp LysLeu Glu Phe Met Phe Arg Leu 145 150 155 160 tat gac acg gat ggg aat ggcttc ctg gac agc tcg gag cta gaa aat 528 Tyr Asp Thr Asp Gly Asn Gly PheLeu Asp Ser Ser Glu Leu Glu Asn 165 170 175 atc atc agt cag atg atg catgtt gca gaa tac ctt gag tgg gat gtc 576 Ile Ile Ser Gln Met Met His ValAla Glu Tyr Leu Glu Trp Asp Val 180 185 190 act gaa ctt aat cca atc ctccat gaa atg atg gaa gaa att gac tat 624 Thr Glu Leu Asn Pro Ile Leu HisGlu Met Met Glu Glu Ile Asp Tyr 195 200 205 gat cat gat gga acc gtg tctctg gag gaa tgg att caa gga gga atg 672 Asp His Asp Gly Thr Val Ser LeuGlu Glu Trp Ile Gln Gly Gly Met 210 215 220 aca acg att cca ctt ctt gtgctc ctg ggc tta gaa aat aac gtg aag 720 Thr Thr Ile Pro Leu Leu Val LeuLeu Gly Leu Glu Asn Asn Val Lys 225 230 235 240 gat gat gga cag cac gtgtgg cga ctg aag cac ttt aac aaa cct gcc 768 Asp Asp Gly Gln His Val TrpArg Leu Lys His Phe Asn Lys Pro Ala 245 250 255 tat tgc aac ctt tgc ctgaac atg ctg att ggc gtg ggg aag cag ggc 816 Tyr Cys Asn Leu Cys Leu AsnMet Leu Ile Gly Val Gly Lys Gln Gly 260 265 270 ctc tgc tgt tcc ttc tgcaag tac aca gtc cat gag cgc tgt gtg gct 864 Leu Cys Cys Ser Phe Cys LysTyr Thr Val His Glu Arg Cys Val Ala 275 280 285 cga gca cct ccc tct tgcatc aag acc tat gtg aag tcc aaa agg aac 912 Arg Ala Pro Pro Ser Cys IleLys Thr Tyr Val Lys Ser Lys Arg Asn 290 295 300 act gat gtc atg cac cattac tgg gtt gaa ggt aac tgc cca acc aag 960 Thr Asp Val Met His His TyrTrp Val Glu Gly Asn Cys Pro Thr Lys 305 310 315 320 tgt gat aag tgc cacaaa act gtt aaa tgt tac cag ggc ctg aca gga 1008 Cys Asp Lys Cys His LysThr Val Lys Cys Tyr Gln Gly Leu Thr Gly 325 330 335 ctg cat tgt gtt tggtgt cag atc aca ctg cat aat aaa tgt gct tct 1056 Leu His Cys Val Trp CysGln Ile Thr Leu His Asn Lys Cys Ala Ser 340 345 350 cat cta aaa cct gaatgt gac tgt gga cct ttg aag gac cat att tta 1104 His Leu Lys Pro Glu CysAsp Cys Gly Pro Leu Lys Asp His Ile Leu 355 360 365 cca ccc aca aca atctgt cca gtg gta ctg cag act ctg ccc act tca 1152 Pro Pro Thr Thr Ile CysPro Val Val Leu Gln Thr Leu Pro Thr Ser 370 375 380 gga gtt tca gtt cctgag gaa aga caa tca aca gtg aaa aag gaa aag 1200 Gly Val Ser Val Pro GluGlu Arg Gln Ser Thr Val Lys Lys Glu Lys 385 390 395 400 agt ggt tcc cagcag cca aac aaa gtg att gac aag aat aaa atg caa 1248 Ser Gly Ser Gln GlnPro Asn Lys Val Ile Asp Lys Asn Lys Met Gln 405 410 415 aga gcc aac tctgtt act gta gat gga caa ggc ctg cag gtc act cct 1296 Arg Ala Asn Ser ValThr Val Asp Gly Gln Gly Leu Gln Val Thr Pro 420 425 430 gtg cct ggt actcac cca ctt tta gtt ttt gtg aac ccc aaa agt ggt 1344 Val Pro Gly Thr HisPro Leu Leu Val Phe Val Asn Pro Lys Ser Gly 435 440 445 gga aaa caa ggagaa cga att tac aga aaa ttc cag tat cta tta aat 1392 Gly Lys Gln Gly GluArg Ile Tyr Arg Lys Phe Gln Tyr Leu Leu Asn 450 455 460 cct cgt cag gtttac agt ctt tct gga aat gga cca atg cca ggg tta 1440 Pro Arg Gln Val TyrSer Leu Ser Gly Asn Gly Pro Met Pro Gly Leu 465 470 475 480 aac ttt ttccgt gat gtt cct gac ttc aga gtg tta gcc tgt ggt gga 1488 Asn Phe Phe ArgAsp Val Pro Asp Phe Arg Val Leu Ala Cys Gly Gly 485 490 495 gat gga accgtg ggc tgg gtt ttg gat tgc ata gaa aag gcc aat gta 1536 Asp Gly Thr ValGly Trp Val Leu Asp Cys Ile Glu Lys Ala Asn Val 500 505 510 ggc aag catcct cca gtt gcg att ctg cct ctt ggg act ggc aat gat 1584 Gly Lys His ProPro Val Ala Ile Leu Pro Leu Gly Thr Gly Asn Asp 515 520 525 cta gca agatgc ctg cga tgg gga gga ggt tac gaa ggt gag aat ctg 1632 Leu Ala Arg CysLeu Arg Trp Gly Gly Gly Tyr Glu Gly Glu Asn Leu 530 535 540 atg aaa attcta aaa gac att gaa aac agc aca gaa atc atg ttg gac 1680 Met Lys Ile LeuLys Asp Ile Glu Asn Ser Thr Glu Ile Met Leu Asp 545 550 555 560 agg tggaag ttt gaa gtc ata cct aat gac aaa gat gag aaa gga gac 1728 Arg Trp LysPhe Glu Val Ile Pro Asn Asp Lys Asp Glu Lys Gly Asp 565 570 575 cca gtgcct tac agt atc atc aat aat tac ttt tcc att ggc gtg gat 1776 Pro Val ProTyr Ser Ile Ile Asn Asn Tyr Phe Ser Ile Gly Val Asp 580 585 590 gcc tccatt gca cac aga ttc cac atc atg aga gaa aaa cac cca gag 1824 Ala Ser IleAla His Arg Phe His Ile Met Arg Glu Lys His Pro Glu 595 600 605 aaa ttcaac agt aga atg aag aac aaa ttt tgg tat ttt gag ttt ggc 1872 Lys Phe AsnSer Arg Met Lys Asn Lys Phe Trp Tyr Phe Glu Phe Gly 610 615 620 aca tctgaa act ttc tca gcc acc tgc aag aag cta cat gaa tct gta 1920 Thr Ser GluThr Phe Ser Ala Thr Cys Lys Lys Leu His Glu Ser Val 625 630 635 640 gaaata gaa tgt gat gga gta cag ata gat tta ata aac atc tct ctg 1968 Glu IleGlu Cys Asp Gly Val Gln Ile Asp Leu Ile Asn Ile Ser Leu 645 650 655 gaagga att gct att ttg aat ata cca agc atg cat gga gga tcc aat 2016 Glu GlyIle Ala Ile Leu Asn Ile Pro Ser Met His Gly Gly Ser Asn 660 665 670 ctttgg gga gag tct aag aaa aga cga agc cat cga cga ata gag aaa 2064 Leu TrpGly Glu Ser Lys Lys Arg Arg Ser His Arg Arg Ile Glu Lys 675 680 685 aaaggg tct gac aaa agg acc acc gtc aca gat gcc aaa gag ttg aag 2112 Lys GlySer Asp Lys Arg Thr Thr Val Thr Asp Ala Lys Glu Leu Lys 690 695 700 tttgca agt caa gat ctc agt gac cag ctg ctg gag gtg gtc ggc ttg 2160 Phe AlaSer Gln Asp Leu Ser Asp Gln Leu Leu Glu Val Val Gly Leu 705 710 715 720gaa gga gcc atg gag atg ggg caa ata tac aca ggc ctg aaa agt gct 2208 GluGly Ala Met Glu Met Gly Gln Ile Tyr Thr Gly Leu Lys Ser Ala 725 730 735ggc cgg cgg ctg gct cag tgc tcc tgc gtg gtc atc agg acg agc aag 2256 GlyArg Arg Leu Ala Gln Cys Ser Cys Val Val Ile Arg Thr Ser Lys 740 745 750tct ctg cca atg caa att gat ggg gag cca tgg atg cag acc cca tgc 2304 SerLeu Pro Met Gln Ile Asp Gly Glu Pro Trp Met Gln Thr Pro Cys 755 760 765aca gtg agt aca gag tag ttgatatgct atgtcaatct cagttttgct 2352 Thr ValSer Thr Glu 770 ttcctctttg actaaataac cacaataact gatttttttc tttatttcttttcaacctat 2412 cagcaaatag tctttttgtt gttgttgtta tgtgtgtgtc agagccactacatttaggct 2472 gtagacatta tatacccttg gcaatgattt agctcttgaa tgtttgtgctagcctaagta 2532 taaatagatc ttttaaatag atcaattata aaccatagat caattataaactatggagct 2592 aaacaaaata ttaataaaag tttatctgaa acttttttgt ttatttcagagcacattatt 2652 agaatattat ttgcgagaaa tgcagaccta agcttatatg tgaacttatttctcagcttt 2712 tctatgcctc catttgggga tttgagggct ttcttctcca taagaaaaaaatttctctcc 2772 agtttctacc ataattaatt gtgttttcca gaatgaggta ttatttaaggcagacactgc 2832 ccctctcaaa aaaaatcagt tttcatttgc atagtgaata ttttattgcatttcaaaaac 2892 atgctaggaa ctgcttttgg cactgggagt agacacatga acaagaccaacagtgtaatt 2952 tccttcaagt tacttacatt cctataatag aggaccgaat aaataaacaactacatgata 3012 aatataactt cagactgtga gagttattaa aaaataaggt gaaatgatgataagaagctg 3072 gattaggtgt ggagaataaa tactacttga gataagggag acctctttgaaaggacatag 3132 ccaaaagctt agtataaaat taaaaaaaat aaaaaaaaaa 3172 5 773PRT Homo sapiens 5 Met Thr Asn Gln Glu Lys Trp Ala His Leu Ser Pro SerGlu Phe Ser 1 5 10 15 Gln Leu Gln Lys Tyr Ala Glu Tyr Ser Thr Lys LysLeu Lys Asp Val 20 25 30 Leu Glu Glu Phe His Gly Asn Gly Val Leu Ala LysTyr Asn Pro Glu 35 40 45 Gly Lys Gln Asp Ile Leu Asn Gln Thr Ile Asp PheGlu Gly Phe Lys 50 55 60 Leu Phe Met Lys Thr Phe Leu Glu Ala Glu Leu ProAsp Asp Phe Thr 65 70 75 80 Ala His Leu Phe Met Ser Phe Ser Asn Lys PhePro His Ser Ser Pro 85 90 95 Met Val Lys Ser Lys Pro Ala Leu Leu Ser GlyGly Leu Arg Met Asn 100 105 110 Lys Gly Ala Ile Thr Pro Pro Arg Thr ThrSer Pro Ala Asn Thr Cys 115 120 125 Ser Pro Glu Val Ile His Leu Lys AspIle Val Cys Tyr Leu Ser Leu 130 135 140 Leu Glu Arg Gly Arg Pro Glu AspLys Leu Glu Phe Met Phe Arg Leu 145 150 155 160 Tyr Asp Thr Asp Gly AsnGly Phe Leu Asp Ser Ser Glu Leu Glu Asn 165 170 175 Ile Ile Ser Gln MetMet His Val Ala Glu Tyr Leu Glu Trp Asp Val 180 185 190 Thr Glu Leu AsnPro Ile Leu His Glu Met Met Glu Glu Ile Asp Tyr 195 200 205 Asp His AspGly Thr Val Ser Leu Glu Glu Trp Ile Gln Gly Gly Met 210 215 220 Thr ThrIle Pro Leu Leu Val Leu Leu Gly Leu Glu Asn Asn Val Lys 225 230 235 240Asp Asp Gly Gln His Val Trp Arg Leu Lys His Phe Asn Lys Pro Ala 245 250255 Tyr Cys Asn Leu Cys Leu Asn Met Leu Ile Gly Val Gly Lys Gln Gly 260265 270 Leu Cys Cys Ser Phe Cys Lys Tyr Thr Val His Glu Arg Cys Val Ala275 280 285 Arg Ala Pro Pro Ser Cys Ile Lys Thr Tyr Val Lys Ser Lys ArgAsn 290 295 300 Thr Asp Val Met His His Tyr Trp Val Glu Gly Asn Cys ProThr Lys 305 310 315 320 Cys Asp Lys Cys His Lys Thr Val Lys Cys Tyr GlnGly Leu Thr Gly 325 330 335 Leu His Cys Val Trp Cys Gln Ile Thr Leu HisAsn Lys Cys Ala Ser 340 345 350 His Leu Lys Pro Glu Cys Asp Cys Gly ProLeu Lys Asp His Ile Leu 355 360 365 Pro Pro Thr Thr Ile Cys Pro Val ValLeu Gln Thr Leu Pro Thr Ser 370 375 380 Gly Val Ser Val Pro Glu Glu ArgGln Ser Thr Val Lys Lys Glu Lys 385 390 395 400 Ser Gly Ser Gln Gln ProAsn Lys Val Ile Asp Lys Asn Lys Met Gln 405 410 415 Arg Ala Asn Ser ValThr Val Asp Gly Gln Gly Leu Gln Val Thr Pro 420 425 430 Val Pro Gly ThrHis Pro Leu Leu Val Phe Val Asn Pro Lys Ser Gly 435 440 445 Gly Lys GlnGly Glu Arg Ile Tyr Arg Lys Phe Gln Tyr Leu Leu Asn 450 455 460 Pro ArgGln Val Tyr Ser Leu Ser Gly Asn Gly Pro Met Pro Gly Leu 465 470 475 480Asn Phe Phe Arg Asp Val Pro Asp Phe Arg Val Leu Ala Cys Gly Gly 485 490495 Asp Gly Thr Val Gly Trp Val Leu Asp Cys Ile Glu Lys Ala Asn Val 500505 510 Gly Lys His Pro Pro Val Ala Ile Leu Pro Leu Gly Thr Gly Asn Asp515 520 525 Leu Ala Arg Cys Leu Arg Trp Gly Gly Gly Tyr Glu Gly Glu AsnLeu 530 535 540 Met Lys Ile Leu Lys Asp Ile Glu Asn Ser Thr Glu Ile MetLeu Asp 545 550 555 560 Arg Trp Lys Phe Glu Val Ile Pro Asn Asp Lys AspGlu Lys Gly Asp 565 570 575 Pro Val Pro Tyr Ser Ile Ile Asn Asn Tyr PheSer Ile Gly Val Asp 580 585 590 Ala Ser Ile Ala His Arg Phe His Ile MetArg Glu Lys His Pro Glu 595 600 605 Lys Phe Asn Ser Arg Met Lys Asn LysPhe Trp Tyr Phe Glu Phe Gly 610 615 620 Thr Ser Glu Thr Phe Ser Ala ThrCys Lys Lys Leu His Glu Ser Val 625 630 635 640 Glu Ile Glu Cys Asp GlyVal Gln Ile Asp Leu Ile Asn Ile Ser Leu 645 650 655 Glu Gly Ile Ala IleLeu Asn Ile Pro Ser Met His Gly Gly Ser Asn 660 665 670 Leu Trp Gly GluSer Lys Lys Arg Arg Ser His Arg Arg Ile Glu Lys 675 680 685 Lys Gly SerAsp Lys Arg Thr Thr Val Thr Asp Ala Lys Glu Leu Lys 690 695 700 Phe AlaSer Gln Asp Leu Ser Asp Gln Leu Leu Glu Val Val Gly Leu 705 710 715 720Glu Gly Ala Met Glu Met Gly Gln Ile Tyr Thr Gly Leu Lys Ser Ala 725 730735 Gly Arg Arg Leu Ala Gln Cys Ser Cys Val Val Ile Arg Thr Ser Lys 740745 750 Ser Leu Pro Met Gln Ile Asp Gly Glu Pro Trp Met Gln Thr Pro Cys755 760 765 Thr Val Ser Thr Glu 770 6 3172 DNA Homo sapiens 6 ttttttttttatttttttta attttatact aagcttttgg ctatgtcctt tcaaagaggt 60 ctcccttatctcaagtagta tttattctcc acacctaatc cagcttctta tcatcatttc 120 accttattttttaataactc tcacagtctg aagttatatt tatcatgtag ttgtttattt 180 attcggtcctctattatagg aatgtaagta acttgaagga aattacactg ttggtcttgt 240 tcatgtgtctactcccagtg ccaaaagcag ttcctagcat gtttttgaaa tgcaataaaa 300 tattcactatgcaaatgaaa actgattttt tttgagaggg gcagtgtctg ccttaaataa 360 tacctcattctggaaaacac aattaattat ggtagaaact ggagagaaat ttttttctta 420 tggagaagaaagccctcaaa tccccaaatg gaggcataga aaagctgaga aataagttca 480 catataagcttaggtctgca tttctcgcaa ataatattct aataatgtgc tctgaaataa 540 acaaaaaagtttcagataaa cttttattaa tattttgttt agctccatag tttataattg 600 atctatggtttataattgat ctatttaaaa gatctattta tacttaggct agcacaaaca 660 ttcaagagctaaatcattgc caagggtata taatgtctac agcctaaatg tagtggctct 720 gacacacacataacaacaac aacaaaaaga ctatttgctg ataggttgaa aagaaataaa 780 gaaaaaaatcagttattgtg gttatttagt caaagaggaa agcaaaactg agattgacat 840 agcatatcaactactctgta ctcactgtgc atggggtctg catccatggc tccccatcaa 900 tttgcattggcagagacttg ctcgtcctga tgaccacgca ggagcactga gccagccgcc 960 ggccagcacttttcaggcct gtgtatattt gccccatctc catggctcct tccaagccga 1020 ccacctccagcagctggtca ctgagatctt gacttgcaaa cttcaactct ttggcatctg 1080 tgacggtggtccttttgtca gacccttttt tctctattcg tcgatggctt cgtcttttct 1140 tagactctccccaaagattg gatcctccat gcatgcttgg tatattcaaa atagcaattc 1200 cttccagagagatgtttatt aaatctatct gtactccatc acattctatt tctacagatt 1260 catgtagcttcttgcaggtg gctgagaaag tttcagatgt gccaaactca aaataccaaa 1320 atttgttcttcattctactg ttgaatttct ctgggtgttt ttctctcatg atgtggaatc 1380 tgtgtgcaatggaggcatcc acgccaatgg aaaagtaatt attgatgata ctgtaaggca 1440 ctgggtctcctttctcatct ttgtcattag gtatgacttc aaacttccac ctgtccaaca 1500 tgatttctgtgctgttttca atgtctttta gaattttcat cagattctca ccttcgtaac 1560 ctcctccccatcgcaggcat cttgctagat cattgccagt cccaagaggc agaatcgcaa 1620 ctggaggatgcttgcctaca ttggcctttt ctatgcaatc caaaacccag cccacggttc 1680 catctccaccacaggctaac actctgaagt caggaacatc acggaaaaag tttaaccctg 1740 gcattggtccatttccagaa agactgtaaa cctgacgagg atttaataga tactggaatt 1800 ttctgtaaattcgttctcct tgttttccac cacttttggg gttcacaaaa actaaaagtg 1860 ggtgagtaccaggcacagga gtgacctgca ggccttgtcc atctacagta acagagttgg 1920 ctctttgcattttattcttg tcaatcactt tgtttggctg ctgggaacca ctcttttcct 1980 ttttcactgttgattgtctt tcctcaggaa ctgaaactcc tgaagtgggc agagtctgca 2040 gtaccactggacagattgtt gtgggtggta aaatatggtc cttcaaaggt ccacagtcac 2100 attcaggttttagatgagaa gcacatttat tatgcagtgt gatctgacac caaacacaat 2160 gcagtcctgtcaggccctgg taacatttaa cagttttgtg gcacttatca cacttggttg 2220 ggcagttaccttcaacccag taatggtgca tgacatcagt gttccttttg gacttcacat 2280 aggtcttgatgcaagaggga ggtgctcgag ccacacagcg ctcatggact gtgtacttgc 2340 agaaggaacagcagaggccc tgcttcccca cgccaatcag catgttcagg caaaggttgc 2400 aataggcaggtttgttaaag tgcttcagtc gccacacgtg ctgtccatca tccttcacgt 2460 tattttctaagcccaggagc acaagaagtg gaatcgttgt cattcctcct tgaatccatt 2520 cctccagagacacggttcca tcatgatcat agtcaatttc ttccatcatt tcatggagga 2580 ttggattaagttcagtgaca tcccactcaa ggtattctgc aacatgcatc atctgactga 2640 tgatattttctagctccgag ctgtccagga agccattccc atccgtgtca taaaggcgaa 2700 acataaactcaagcttatcc tcaggtcttc ctctttcaag cagagacagg taacagacaa 2760 tgtccttcagatggattact tctggggaac acgtatttgc aggagaagta gttcggggag 2820 gggtgatggcacctttattc attctcagac cgcctgatag gagagcaggc ttacttttta 2880 ccattggactagaatgagga aacttgttgc taaatgacat gaaaaggtgt gcagtgaaat 2940 catcaggaagctcggcttcc aggaatgtct tcatgaatag tttgaaacct tcaaaatcta 3000 ttgtttggttaagaatgtct tgtttccctt caggattata ctttgcaagc acaccattac 3060 catggaattcttcaagaaca tcctttaatt tctttgtaga atactcagca tatttctgaa 3120 gttgggaaaattccgaaggg ctgaggtggg cccatttttc ctggtttgtc at 3172 7 801 PRT Rattus sp.7 Met Thr Asn Gln Glu Lys Trp Ala His Leu Ser Pro Ser Glu Phe Ser 1 5 1015 Gln Leu Gln Lys Tyr Ala Glu Tyr Ser Thr Lys Lys Leu Lys Asp Val 20 2530 Leu Glu Glu Phe His Gly Asn Gly Val Leu Ala Lys Tyr Asn Pro Glu 35 4045 Gly Lys Gln Asp Ile Leu Asn Gln Thr Ile Asp Phe Glu Gly Phe Lys 50 5560 Leu Phe Met Lys Thr Phe Leu Glu Ala Glu Leu Pro Asp Asp Phe Thr 65 7075 80 Ala His Leu Phe Met Ser Phe Ser Asn Lys Phe Pro His Ser Ser Pro 8590 95 Asn Val Lys Ser Lys Pro Ala Leu Leu Ser Gly Gly Leu Arg Met Asn100 105 110 Lys Gly Ala Ile Thr Pro Pro Arg Ser Ser Pro Ala Asn Thr CysPhe 115 120 125 Pro Glu Val Ile His Leu Lys Asp Ile Val Cys Tyr Leu SerLeu Leu 130 135 140 Glu Arg Gly Arg Pro Glu Asp Lys Leu Glu Phe Met PheArg Leu Tyr 145 150 155 160 Asp Thr Asp Gly Asn Gly Phe Leu Asp Ser SerGlu Leu Glu Asn Ile 165 170 175 Ile Gly Gln Met Met His Val Ala Glu TyrLeu Glu Trp Asp Val Thr 180 185 190 Glu Leu Asn Pro Ile Leu His Glu MetMet Glu Glu Ile Asp Tyr Asp 195 200 205 Arg Asp Gly Thr Val Ser Leu GluGlu Trp Ile Gln Gly Gly Met Thr 210 215 220 Thr Ile Pro Leu Leu Val LeuLeu Gly Leu Glu Asn Asn Val Lys Asp 225 230 235 240 Asp Gly Gln His ValTrp Arg Leu Lys His Phe Asn Lys Pro Ala Tyr 245 250 255 Cys Asn Leu CysLeu Asn Met Leu Ile Gly Val Gly Lys Gln Gly Leu 260 265 270 Cys Cys SerPhe Cys Lys Tyr Thr Val His Glu Arg Cys Ala Arg Ala 275 280 285 Pro ProSer Cys Ile Lys Thr Tyr Val Lys Ser Lys Lys Asn Thr Asp 290 295 300 ValMet His His Tyr Trp Val Glu Gly Asn Cys Pro Thr Lys Cys Asp 305 310 315320 Lys Cys His Lys Thr Val Lys Cys Tyr Gln Gly Leu Thr Gly Leu His 325330 335 Cys Val Trp Cys Gln Thr Thr Leu His Asn Lys Cys Ala Ser His Leu340 345 350 Lys Pro Glu Cys Asp Cys Gly Pro Leu Lys Asp His Ile Leu ProPro 355 360 365 Thr Thr Ile Cys Pro Val Val Leu Thr Met Pro Thr Ala GlyThr Ser 370 375 380 Val Pro Glu Glu Arg Gln Ser Thr Ala Lys Lys Glu LysGly Ser Ser 385 390 395 400 Gln Gln Pro Asn Lys Val Thr Asp Lys Asn LysMet Gln Arg Ala Asn 405 410 415 Ser Val Thr Met Asp Gly Gln Gly Leu GlnIle Thr Pro Ile Pro Gly 420 425 430 Thr His Pro Leu Leu Val Phe Val AsnPro Lys Ser Gly Gly Lys Gln 435 440 445 Gly Glu Arg Ile Tyr Arg Lys PheGln Tyr Leu Leu Asn Pro Arg Gln 450 455 460 Val Tyr Ser Leu Ser Gly AsnGly Pro Met Pro Gly Leu His Phe Phe 465 470 475 480 Arg Asp Val Pro AspPhe Arg Val Leu Ala Cys Gly Gly Asp Gly Thr 485 490 495 Val Gly Trp IleLeu Asp Cys Ile Glu Lys Ala Asn Val Val Lys His 500 505 510 Pro Pro ValAla Ile Leu Pro Leu Gly Thr Gly Asn Asp Leu Ala Arg 515 520 525 Cys LeuArg Trp Gly Gly Gly Tyr Glu Gly Glu Asn Leu Met Lys Ile 530 535 540 LeuLys Asp Ile Glu Ser Ser Thr Glu Ile Met Leu Asp Arg Trp Lys 545 550 555560 Phe Glu Val Thr Pro Asn Asp Lys Asp Glu Lys Gly Asp Pro Val Pro 565570 575 Tyr Ser Ile Ile Asn Asn Tyr Phe Ser Ile Gly Val Asp Ala Ser Ile580 585 590 Ala His Arg Phe His Ile Met Arg Glu Lys His Pro Glu Lys PheAsn 595 600 605 Ser Arg Met Lys Asn Lys Phe Trp Tyr Phe Glu Phe Gly ThrSer Glu 610 615 620 Thr Phe Ser Ala Thr Cys Lys Lys Leu His Glu Ser ValGlu Ile Glu 625 630 635 640 Cys Asp Gly Val Gln Ile Asp Leu Ile Asn IleSer Leu Gln Gly Ile 645 650 655 Ala Ile Leu Asn Ile Pro Ser Met His GlyGly Ser Asn Leu Trp Gly 660 665 670 Glu Ser Lys Lys Lys Arg Ser His ArgArg Ile Glu Lys Lys Gly Ser 675 680 685 Asp Lys Arg Pro Thr Leu Thr AspAla Lys Glu Leu Lys Phe Ala Ser 690 695 700 Gln Asp Leu Ser Asp Gln LeuLeu Glu Val Val Gly Leu Glu Gly Ala 705 710 715 720 Met Glu Met Gly GlnIle Tyr Thr Gly Leu Lys Ser Ala Gly Arg Arg 725 730 735 Leu Ala Gln CysSer Ser Val Val Ile Arg Thr Ser Lys Ser Leu Pro 740 745 750 Met Gln IleAsp Gly Glu Pro Trp Met Gln Thr Pro Cys Thr Ile Lys 755 760 765 Ile ThrHis Lys Asn Gln Ala Pro Met Leu Met Gly Pro Pro Pro Lys 770 775 780 ThrGly Leu Phe Cys Ser Leu Ile Lys Arg Thr Arg Asn Arg Ser Lys 785 790 795800 Glu

What is claimed is:
 1. An isolated human diacylglcerol kinase β (hDAGKβ)protein comprising the amino acid sequence encoded by SEQ ID NO:4.
 2. Amethod for identifying a compound with DAGK modulating activity,comprising contacting a DAGK protein encoded by SEQ ID NO:4 with a testcompound and detecting modulation of enzyme activity or detecting enzymeinactivity.
 3. A method of producing an hDAGKβ protein comprisingintroducing a vector comprising a nucleotide sequence encoding a hDAGKβprotein of claim 1 into a cell, and culturing said cell under conditionssuitable for expression of said hDAGKβ protein.